Nonhuman model animal of th2-mediated hyperimmune response

ABSTRACT

An object of the present invention is to provide a nonhuman model animal of Th2-mediated hyperimmune response lacking PIR-B gene function on its chromosome by which the Th2-mediated immune response mechanism and allergy onset mechanism in vivo can be analyzed and which is liable to suffer from not only hyper-response of B cells but also allergy, and an inducer/promoter or an inhibitor for Th2-mediated immune response, etc. with the use of the nonhuman model animal of Th2-mediated hyperimmune response. The nonhuman model animal of Th2-mediated hyperimmune response is prepared by integrating a fragment comprising exons 1 to 7 and the domain in the 5′ side of exon 8 of mouse PIR-B gene and another fragment containing exons 10 to 14 into a vector pMC1-Neo, cleaving it with Xho I-Sal I, integrating it into a vector pIC19R-MC1tk having herpes virus thymidine kinase to thereby construct targeting vector, transferring the targeting vector into ES cells and then injecting the ES cells into blastcyst.

TECHNICAL FIELD

The present invention relates to a nonhuman model animal of Th2-mediatedhyperimmune response lacking the function of PIR-B gene on itschromosome, which is an inhibitory receptor gene of pairedimmunoglobulin (Ig)-like receptor family liable to suffer from not onlyhyper-response of B cells but also allergy. Further, the presentinvention relates to a method for screening an inducer/promoter or aninhibitor for Th2-mediated immune response or a promoter or an inhibitorfor PIR-A function, and a method for screening a therapeutic agent forallergosis.

BACKGROUND ART

T cells controlling humoral immunity and cellular immunity are mostlyCD4⁺T cells, which can be classified into Th (helper T cells) 1 or Th2cells respectively, by patterns for producing cytokine. Th1 cells areknown to produce IFN-γ and IL (interleukin) −2 and induce activation ofmacrophage, while Th2 cells are known to promote activation and antibodygeneration of B cells by secreting IL-4, IL-5, IL-10, IL-13 and thelike. Currently, the studies related to differentiation of Th1 and Th2has gathered attention, since they are thought to be deeply involved inthe cause of immune mediated illness. Th1 has been suggested itsrelevance to delayed-type allergy and organ-specific autoimmune disease,whereas Th2 has been suggested its relevance to type 1 allergy andsystemic autoimmune disease (SLE and others). However, because they arein antagonistic relationship each other, these diseases are expectedthat they can be prevented or treated by controlling Th1 or Th2. Indeed,it has been reported that administering IL-12 with an antigen forinfectious disease such as leishmania induces Th1 preventing developmentto forfend infection.

On the other hand, PIR of mice whose expression is found in B cells,mast cells, dendritic cells and macrophages are type 1transmembrane-type glycoprotein, belonging to immunoglobulin (Ig)-likereceptor family, have 6 Ig domains extracellularly and they areclassified into two subtypes of PIR-A and PIR-B by their differences ofintracellular structure. The immunoglobulin (Ig)-like receptors PIR-Aand PIR-B (J. Biol. Chem. 272, 7320-7327, 1997; Proc. Natl. Acad. Sci.USA. 94, 5261-5266, 1997) have been known to be the activating-type orinhibitory receptors of Ig-like receptor (IgLR) family expressing in apair-wise fashion on a wide variety of cells mostly in the immune system(Science 290, 84-89, 2000). It has been reported that aforementionedPIR-A requires Fc receptor γ chain for its expression on cell surfaceand for delivery of activation signaling (J. Exp. Med. 188, 991-995,1998; J. Exp. Med. 189, 309-318, 1999; J. Immunol. 161, 4042-4047, 1998;J. Biol. Chem. 274, 30288-30296, 1999). In contrast, PIR-B has beenreported that it contains immunoreceptor tyrosine-based inhibitorymotifs (ITIMs) in its cytoplasmic portion and inhibits receptor-mediatedactivation signaling in vitro by engaging antigen receptor (BCR) on Bcells and other activating-type receptors (J. Immunol. 161, 4042-4047,1998; Proc. Natl. Acad. Sci. USA. 95, 2446-2451, 1998; J. Exp. Med. 187,1355-1360, 1998). However, neither the physiological function nor ligandfor PIR has been elucidated yet.

An object of the present invention is to provide a nonhuman model animalof Th2-mediated hyperimmune response lacking PIR-B gene function on itschromosome by which the Th2-mediated immune response mechanism andallergy onset mechanism in vivo can be analyzed and which is liable tosuffer from not only hyper-response of B cells but also allergy, and toprovide a method for screening an inducer/promoter or an inhibitor forTh2-mediated immune response, or a promoter or an inhibitor for PIR-Afunction, a method for screening a therapeutic agent for allergosis anda method for diagnosing allergosis with the use of the nonhuman modelanimal of Th2 -medeated hyperimmune response.

The present inventors have made a keen study to elucidate physiologicalfunction for PIR-B being an inhibitory receptor pairs in the pairedimmunoglobulin-like receptor family, and have found that the micelacking PIR-B gene function on its chromosome show Th2-prone humoralresponses which are liable to suffer from not only hyper-response of Bcells but also allergy to complete the present invention. That is, thepresent invention has completed based on the findings as follows.Examining above-mentioned PIR-B-deficient (PIR-B^(−/−)) mice revealedthat they have increased the number of peritoneal B-1 cells and that theB-1 cells and splenic B-2 cells of the PIR-B-^(−/−) mice showedhypersensitivity to BCR. The nonhuman model animal of Th2-mediatedhyperimmune response has been constructed based on this knowledge. Inaddition, immunizing PIR-B-^(−/−) mice with T-independent (TI) antigensshowed the enhanced IgM response compared with wild-type mice. It isthus found that PIR-B comprises an inhibitory ability for activationsignaling via BCR under physiological conditions and it candown-regulate the size of the B-1 cell population. Moreover, immunizingPIR-B^(−/−) mice with TD antigens (an alum adjuvant mixed with TNP-KLHwhich initiates Th2-type response and pertussis toxin) allowed thepresent inventors to find that the antibody titer of IgG1 was especiallyrose and IL-4 production in PIR-B-deficient mice was dominantlyincreased compared to wild-type mice. From these points of view, it hasbeen shown that PIR-B-deficient mice exhibit Th-2-type prone humoralresponse, which might be caused by the impaired maturation of dendriticcells (DCs) in PIR-B^(−/−) mice. Furthermore, in the present invention,based on this knowledge, a method for screening an inducer/promote or aninhibitor for Th2-mediated immune response or a promoter or an inhibitorfor PIR-A function, a method for screening a therapeutic agent forallergosis, a method for diagnosis of allergosis and the like have beenconstructed.

DISCLOSURE OF THE INVENTION

The present invention relates to a nonhuman model animal of Th2-mediatedhyperimmune response which induces hyper-response of a B cell andfacilitates development of an allergy by lacking PIR-B gene function onits chromosome (“1”); the nonhuman model animal of Th2-mediatedhyperimmune response according to “1”, wherein it develops the allergymore excessively than a wild-type nonhuman animal by immunizing anadjuvant that initiates Th2-mediated response (“2”); the nonhuman modelanimal of Th2 -mediated hyperimmune response according to “2”, whereinthe adjuvant that initiates Th2 -mediated response is an alum adjuvantcomprising a TNP-KLH and a pertussis toxin (“3”); and the nonhuman modelanimal of Th2-mediated hyperimmune response according to any one of “1”to “3”, wherein the nonhuman animal is a mouse (“4”).

The present invention also relates to a method for screening aninducer/promoter or an inhibitor for Th2-mediated immune response or apromoter or an inhibitor for PIR-A function, wherein an alteration ofTh2-mediated immune response or PIR-A function is determined/evaluatedby administering a test substance to the nonhuman model animal ofTh2-mediated hyperimmune response according to any one of “1” to “4” orcontacting a tissue, an organ or a cell derived from the animal to thetest substance (“5”) ; the method for screening an inducer/promoter oran inhibitor for Th2-mediated immune response or a promoter or aninhibitor for PIR-A function according to “5”, wherein the alteration ofTh2-mediated immune response is an alteration of activation of the Bcell, generation of antibody, production of cytokine and/or antigenpresentational ability in an antigen presenting cell (“6”) ; the methodfor screening an inducer/promoter or an inhibitor for Th2-mediatedimmune response or a promoter or an inhibitor for PIR-A functionaccording to “6”, wherein the alteration of activation of the B cell isan alteration of development or proliferation of the B cell (“7”); themethod for screening an inducer/promoter or an inhibitor forTh2-mediated immune response or a promoter or an inhibitor for PIR-Afunction according to “6”, wherein the alteration of generation ofantibody is an alteration of generation of an IgM antibody, an IgG1antibody, an IgG2b antibody and/or an IgG2a antibody (“8”); the methodfor screening an inducer/promoter or an inhibitor for Th2-mediatedimmune response or a promoter or an inhibitor for PIR-A functionaccording to “6”, wherein the alteration of production of cytokine is analteration of production of interleukin-4, IFN-γ and/or interleukin-12(“9”); the method for screening an inducer/promoter or an inhibitor forTh2-mediated immune response or a promoter or an inhibitor for PIR-Afunction according to “6”, wherein the alteration of antigenpresentational ability in the antigen presenting cell is an alterationof expression amount of a major histocompatibility complex class II,CD80 and/or CD 86 in a dendritic cell (“10”); and the method forscreening an inducer/promoter or an inhibitor for Th2-mediated immuneresponse or a promoter or an inhibitor for PIR-A function according toany one of “5” to “10”, wherein a case with the nonhuman model animal ofTh2-mediated hyperimmune response according to any one of “1” to “4” iscompared/evaluated with a case with the wild-type nonhuman animal(“11”).

The present invention further relates to a method for screening atherapeutic agent for an allergosis, wherein the alteration ofTh2-mediated immune response or PIR-A or PIR-B function isdetermined/evaluated by administering a Th2-mediated immune responseinitiating adjuvant and the test substance to the nonhuman model animalof Th2-mediated hyperimmune response according to any one of “1” to “4”,or contacting the tissue, the organ or the cell derived from the animalto the Th2-mediated immune response initiating alum adjuvant and thetest substance (“12”); the method for screening a therapeutic agent foran allergosis wherein the alteration of Th2-mediated immune response isan alteration of activation of the B cell, generation of antibody,production of cytokine and/or antigen presentational ability in theantigen presenting cell (“13”); the method for screening a therapeuticagent for an allergosis according to “13”, wherein the alteration of theB cell is an alteration of development or proliferation of the B cell(“14”); the method for screening a therapeutic agent for an allergosisaccording to “13”, wherein the alteration of generation of antibody isan alteration of generation of an IgM antibody, an IgG1 antibody, anIgG2b antibody and/or an IgG2a antibody (“15”); the method for screeninga therapeutic agent for an allergosis according to “13”, wherein thealteration of production of cytokine is an alteration of production ofinterleukin-4, IFN-γ and/or interleukin-12 (“16”); and the method forscreening a therapeutic agent for an allergosis according to “13”,wherein the alteration of antigen presentational ability in the antigenpresenting cell is an alteration of expression amount of the majorhistocompatibility complex class II, CD80 and/or CD 86 in the dendriticcell (“17”).

The present invention still further relates to an inducer/promoter or aninhibitor for Th2-mediated immune response or a promoter or an inhibitorfor PIR-A function obtained by the method for screening aninducer/promoter or an inhibitor for Th2-mediated immune response or apromoter or an inhibitor for PIR-A or PIR-B function according to anyone of “5” to “11” (“18”); the inducer/promoter or the inhibitor forTh2-mediated immune response or the promoter or the inhibitor for PIR-Afunction according to “18”, wherein the inducer/promoter or theinhibitor for Th2 -mediated immune response is a therapeutic agent for adisorder attributed to Th2 -mediated hyperimmune response (“19”); theinducer/promoter or the inhibitor for Th2-mediated immune response orthe promoter or the inhibitor for PIR-A function according to “19”,wherein the disorder attributed to Th2-mediated hyperimmune response isan allergosis (“20”); a therapeutic agent for an allergosis obtained bythe method for screening a therapeutic agent for an allergosis accordingto any one of “12” to “17” (“21”); a method for diagnosis of anallergosis wherein a disease symptom of the nonhuman model animal ofTh2-mediated hyperimmune response according to any one of “1” to “4” isutilized therefor (“22”).

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows graphs indicating a gene map of PIR-B knockout mice of thepresent invention and wild-type mice (a), the result of Southern blotanalysis in each mouse (b), the result of immunoblot analysis (c) andthe result of flow cytometric analysis of expression of PIR on cellsurface of BM-macrophages, BM-DCs, splenic B cells, and BM-MCs,respectively.

(a) Organization of PIR-B gene, construction of the targeting vector(TK: thymidine kinase gene, Neo: neomycin resistance gene), and thestructure of the targeted genome are shown. In the drawing, H, P, RI, RVand S represent the restriction site of Hin dIII, Pst I, Eco RI, Eco RVand Sph I, respectively.

(b) Genomic DNA from homozygotic mice of littermates obtained fromintercrossing heterozygotes was cleaved with Eco RI, and the resultingfragments were subjected to Southern blot analysis with a probe of FIG.1 a (Pst 1-Sph 1 fragment). The positions of 5.6 kb and 4.8 kb representwild-type allele (+) and mutated form allele (−), respectively.

(c) Splenic B cells (2×10⁶ cells) from wild-type mice (+/+) orPIR-B^(−/−) mice (−/−) were subjected to immunoblot analysis using goatanti-PIR polyclonal antibodies. Position of PIR-B of approximately120-kDa is indicated by an arrow.

(d) The cells from wild-type (deep-colored line) or those fromPIR-B^(−/−) mice (light-colored line) were reacted with phycoerythrin(PE)-conjugated anti-mouse PIR-A/B antibody (6C1) or PE-conjugated ratIgG1 monoclonal antibody (dotted line; control), stained withFITC-labeled monoclonal antibodies such as anti-CD11b antibody, antiCD11c antibody, anti B220 antibody, or anti-c-kit antibody to separatepositive cells.

FIG. 2 shows graphs indicating the results of analysis of development ofB cells in PIR-B^(−/−) mice of the present invention and wild-type miceby flow cytometry.

(a) Bone-marrow cells were stained with B220 and IgM, and separated pro,pre, immature and mature B cells by flow cytometric analysis.

(b) Splenic B220 positive cells were stained with IgM and IgD andseparated to show mature IgM^(lo)IgD^(hi) B cells, type 2 transitional Bcells (T-2 B cells) of IgM^(hi)IgD^(hi), and type 1 transitional B cells(T-1 B cells) of IgM^(hi)IgD^(lo).

(c) Splenic B220 positive cells were stained with CD21 and CD23 andseparated to show newly formed CD21⁻CD23⁻ B cells, CD21⁺CD23⁺ B cellsand CD21⁺CD23⁻ B cells.

(d) Peritoneal cells were stained with CD5 and IgM, and separatedleukocytes to show CD5⁺IgM⁺B-1 cells.

The numbers near fractions in each of the graphs represent the ratios ofcells existing in fractions in all cells. The result of each panelindicates the mean obtained from three independent experiments.

FIG. 3 shows graphs indicating the results of proliferative responses ofB cells derived from PIR-B-deficient mice of the present invention toBCR stimulation.

(a) B220 positive splenic B cells obtained from wild-type mice (closedcolumn) and those from PIR-B^(−/−) mice (open column) were cultured inthe coexistence of anti-μ F (ab′)₂ antibody at each of the concentrationindicated in the graph, 1 μg/ml of anti-CD40 antibody, or 10 μg/ml ofLPS (positive control). The respective results from triplicateexperiments are shown as mean values±s.d.

(b) B220 posotive splenic B cells were stimulated with 10 μg/ml ofanti-μ antibody in the presence or absence of 25 μg/ml of 2.4G2 toinhibit Fcγ receptor IIB.

(c) Wild-type mice (closed column) or PIR-B^(−/−) mice (open column)were injected with 200 μg of anti-μ (ab′)₂ antibody intraperitoneally.Before or after 48 h of the injection, ratio of B-1 cell population inperitoneal lymphocytes was estimated by flow cytometry with CD5 and IgMstaining. The respective results from triplicate experiments are shownas mean values±s.d. While B-1 cells from wild-type did not respond,those from PIR-B^(−/−) mice proliferated by the anti-μ antibodystimulation. In the graphs, “*”, “**”, and N.S. represent p<0.05,p<0.01, and not significant, respectively.

FIG. 4 shows graphs indicating the results of examination of humoralresponse to T1 antigens in PIR-B^(−/−) mice of the present invention orwild-type mice.

Wild-type and PIR-B^(−/−) mice at 8 weeks of age were immunized with 100μg of TNP-Ficoli (a) or 50 μg of TNP-LPS (b). Relative amounts ofTNP-specific IgM were determined by ELISA. The sera collected beforeimmunization (pre-immune) and the sera collected on the 7th day fromimmunization (immune) were determined with absorbance at 450 nm. In thegraphs, open symbols and closed symbols represent the data ofPIR-B^(−/−) mice and of wild-type mice, respectively, and “*” and “**”represent p<0.05 and p<0.01, respectively.

FIG. 5 shows graphs indicating the results of Th2-mediated immuneresponses to TD antigens in PIR-B^(−/−) mice of the present invention orin wild-type mice.

(a) This shows graphs indicating the result of the examination ofhumoral response to TNP-KLH. In order to measure immune response tothymus-dependent antigens (TD antigens), wild-type mice (closed squares:n=5) or PIR-B^(−/−) mice (open squares: n=5) at 8 weeks of age wereimmunized with the mixture of 10 μg of TNP-KLH and 0.25 μg of pertussistoxin with alum adjuvant intraperitoneally. Further, in order to observethe secondary immune response, mice were injected with the mixture of 1μg of TNP-KLH and 0.25 μg of pertussis toxin with alum adjuvantintraperitoneally at 8th week from the primary immunization. Blood wascollected before and at every 2 weeks after the immunization. The titerof anti-TNP antibody in serum was determined by ELISA. In the graphs,arrowheads represent the points at which mice were immunized (at 0 and 8week). With the use of ELISA, relative amounts of TNP-specific IgM,IgG1, IgG2a and IgGb were determined (absorbance: 450 nm). In thegraphs, “*” represents p<0.05.

(b) This shows graphs indicating enhanced IL-4 production and reducedIFN-y production of lymphocytes from PIR-B^(−/−) mice to antigen. Inorder to examine the response of T-cells in immune response to TDantigens, mice were immunized onto their foodpads with the mixture of 10μg of TNP-KLH (upper panel) or OVA (lower panel) and 0.25 μg ofpertussis toxin with alum adjuvant. After 11 days from the primaryimmunization, popliteal lymph nodes were taken out from wild-type mice(closed column) or PIR-B^(−/−) mice (open column) to collect lymph nodecells, and the lymph node cells from wild-type or PIR-B-deficient micewere re-stimulated with TNP-KLH or OVA. After 40 h from stimulation, theculture supernatant was collected to determine IL-4 or IFN-γ in it byELISA. Proliferation of lymph node cells was determined by[³H]-thymidine uptake on the 3rd day from the culture. Mean values(±s.d.) of triplicate cultures are shown as data. Two independentexperiments were conducted to obtain theses results, which were similarto each other. In the figure, “*” and “**” represent p<0.05 and p<0.01,respectively.

FIG. 6 shows graphs indicating the results of impaired maturation of DCsand reduced IL12 production in PIR-B^(−/−) mice of the presentinvention.

(a) This shows graphs indicating the results of examining DC maturation.DCs from wild-type mice (bold line) or PIR-B^(−/−) mice (light-coloredline) were incubated for 24 h in the presence or absence of OVA oranti-CD40 antibody. The above-mentioned DCs were then stained andanalyzed by flow cytometry so as to examine the expression of MHC classII (I-A), CD80, or CD86. In DCs from wild-type mice, increasedexpressions were shown in all three molecules mentioned above,indicating an efficient DC maturation. Whereas, in DCs fromPIR-B-deficient mice, impaired DCs maturation was suggested by declineof 1-A expression level and apparent reduction of CD80 and CD86expression.

(b) This is a graph showing DCs producing IL12p70 in response to OVAduring 24 h-culture. Data is shown as the mean values (±s.d.) oftriplicate cultures. In the graph, “*” represents p<0.05.

(c) This shows graphs indicating DCs from wild-type and PIR-B^(−/−) miceuptaking antigen. DSc were incubated in the coexistence ofFITC-conjugated OVA at 37° C. or 4° C. for 3 h, washed, and then stainedwith PE-conjugated anti-CD11c antibody. FITC-labeled OVA uptake by DCswas monitored by flow cytometry. There was no significant difference inamount of antigen uptake between DCs from wild-type and fromPIR-B-deficient mice.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, a nonhuman model animal of Th2 -mediatedhyperimmune response refers a nonhuman animal which shows hyper-responseof B cells and characteristic Th2-prone humoral responses and which isliable to suffer from allergy and the like, for example a nonhumananimal whose PIR-B (p91A) gene (J. Biol. Chem. 272, 7320-7, 1997; Proc.Natl. Acad. Sci. USA. 94, 5261-6, 1997; J. Biochem. 123, 358-68, 1998)function is deficient on its chromosome or the like can be specificallyexemplified, however, it is not particularly limited to them. Thenonhuman animal develops allergy more excessively than a wild-typenonhuman animal by immunizing adjuvant that initiates Th2-mediatedresponse such as alum adjuvant comprising TNP-KLH and pertussis toxin.The aforementioned nonhuman animal lacking PIR-B gene function on itschromosome refers a nonhuman animal whole or part of whose endogenousgene that encodes PIR-B is inactivated by genetic mutation such asdistraction, defect and replacement and that lost function to expressPIR-B. Additionally, a rodent such as a mouse, a rat, and the like canbe specifically exemplified as the nonhuman animal of the presentinvention, however, it is not particularly limited to them.

The wild-type nonhuman animal of the present invention means an animalof the same species as the above-mentioned nonhuman animal lacking PIR-Bgene function on its chromosome, a littermate animal is preferablyexemplified above all. It is desirable to use PIR-B-deficient type inthese homozygotic nonhuman animals which were born at the expectedMendelian ratios and their littermates of wild-type simultaneously,because precise comparative experiment can be conducted at an individuallevel. As a preferable example of the nonhuman model animal ofTh2-mediated hyperimmune response of the present invention and awild-type nonhuman animal, PIR-B knockout mice and wild-type oflittermates of the PIR-B knockout mice are specifically exemplified,respectively. The case where a nonhuman animal is a mouse is explainedwith examples below.

A mouse lacking PIR-B gene function on its chromosome, i.e., PIR-Bknockout mouse (PIR-B^(−/−)) is generated. PIR-B knockout mouse can begenerated by the method such as described in the article (Nature 379,346-349, 1996). In particular, a PIR-B gene is screened with a genefragment obtained from a mouse genomic library by PCR or other suchmethod, whole or part of the screened PIR-B gene is replaced with amarker gene such as neomycin-resistant gene by technique of DNArecombination, a gene such as a diphtheria toxin A fragment (DT-A) geneor a herpes simplex virus thymidine kinase gene (HSV-tK) is introducedinto 5′-terminal side to construct a targeting vector, the constructedtargeting vector is linearized and introduced into ES cells by a methodof such as electroporation, then the ES cells are homologouslyrecombined, and subsequently the ES cells showing resistance againstantibiotic such as G418 or ganciclovir (GANC) among the recombinant areselected. It is preferable to confirm by Southern blotting etc. whetherthese selected ES cells are object recombinants.

The above-mentioned recombinant ES cell is microinjected into blastocystof mouse, and the blastocyst is transplanted into a uterus of arecipient mouse to generate a chimeric mouse. A heterozygote mouse canbe obtained by intercrossing the chimeric mouse with a wild-type mouse,and PIR-B knockout mouse can be generated by intercrossing theheterozygote mice. In addition, as a method for confirming whether PIR-Bgene in the PIR-B knockout mouse is deficient PIR-B cells on itschromosome, a method for examining with Southern blotting and others byisolating DNA from splenic B cells of the mouse obtained fromabove-mentioned method, and a method for examining with immunoblotanalysis and others by using protein extracted from splenic B cells ofthis mouse, can be exemplified.

The nonhuman model animal of Th2-mediated hyperimmune response of thepresent invention is useful for analyzing the Th2-mediated immuneresponse mechanism and allergy onset mechanism. Because the nonhumanmodel animal of Th2-mediated hyperimmune response lacking PIR-B genefunction of the present invention induces inhibition of Th2-mediatedhyperimmune response and the inhibition of PIR-A function is suggested,the nonhuman model animal of Th2-mediated hyperimmune response of thepresent invention can be used for screening a substance which controlsthese biological responses. That is, using the nonhuman model animal ofTh2-mediated hyperimmune response allows not only to screen aninducer/inhibitor for Th2-mediated immune response such as a substancecomprising the same inhibitory action as PIR-B, an inducer/promoter forTh2-mediated immune response such as a substance promoting Th2-mediatedimmune response induction, a promoter for PIR-A function such as asubstance enhancing PIR-A function which is declined in the nonhumanmodel animal of Th2-mediated hyperimmune response of the presentinvention, an inhibitor for PIR-A function such as a substanceinhibiting PIR-A function further, a therapeutic agent for an allergosisand the like but also to diagnose of an allergosis.

As a method for screening an inducer/promoter or an inhibitor forTh2-mediated immune response or a promoter or an inhibitor for PIR-Afunction, a method for measuring/evaluating the alteration ofTh2-mediated immune response such as alternation of activation of Bcells, generation of antibodies, production of cytokine, antigenpresentational ability in the antigen presenting cells, or PIR-Afunction by administering the test substance to the nonhuman modelanimal of Th2-mediated hyperimmune response of the present invention orcontacting the tissue, the organs or the cells from the nonhuman modelanimal of Th2-mediated hyperimmune response to the test substance can bespecifically exemplified but it is not limited to them. As a method formeasuring/evaluating the alteration of activation of B cells, forexample, a method for measuring/evaluating activation of B cells such asdevelopment of B cells, proliferation of B cells, sensitivity of B cellssuch as B-1 cells or B-2 cells for binding with BCR in condition withand without the test substance to specify a substance in whichalteration has occurred, can be exemplified. As a method formeasuring/evaluating the alteration of generation of antibody, forexample, a method for measuring/evaluating increase or decrease ofgeneration of antibody such as IgM antibody, IgG1 antibody, IgG2bantibody, IgG2a antibody in condition with and without the testsubstance to specify a substance in which alteration has occurred, canbe exemplified. As a method for measuring/evaluating the alteration ofproduction of cytokine, for example, a method for measuring/evaluatingincrease or decrease of production of cytokine such as interleukine-4,IFN-γ, interleukine-12 in condition with and without the test substanceto specify a substance in which alteration has occurred, can beexemplified. Further, as a method for measuring/evaluating thealteration of antigen presentational ability in antigen presentingcells, for example, a method for measuring/evaluating increase ordecrease of expression amount of antigen which is specific to antigenpresenting cells such as a major histocompatibility complex class II,CD80, CD86 and the like in antigen presenting cells such as dendriticcells in condition with and without the test substance to specify asubstance in which alteration has occurred, can be exemplified. Thoughabove-mentioned methods for measuring/evaluating are not limited tothese specifically, in screening an inducer/promoter or an inhibitor forTh2-mediated immune response, or a promoter or an inhibitor for PIR-Afunction, it is more preferable to compare/evaluate the nonhuman modelanimal of Th2-mediated hyperimmune response with the wild-type nonhumananimal of its littermate.

As a method for screening a therapeutic agent for an allergosis, forexample, a method for measuring/evaluating the alteration ofTh2-mediated immune response such as activation of B cells, generationof antibodies, production of cytokine, antigen presentational ability inantigen presenting cells, and the like or PIR-A or PIR-B function byadministering the test substance to the nonhuman model animal ofTh2-mediated hyperimmune response of the present invention or contactingthe tissue, the organs or the cells from the nonhuman model animal ofTh2-mediated hyperimmune response to a test substance to specify thesubstance in which Th2-mediated immune response or PIR-A or PIR-Bfunction is improved, can be exemplified, but it is not limited to thesemethods. As a method for measuring/evaluating the alteration ofactivation of B cells, for example, a method for measuring/evaluatingactivation of B cells such as development of B cells, proliferation of Bcells, sensitivity of B cells such as B-1 cells or B-2 cells for bindingwith BCR in condition with and without the test substance to specify asubstance in which the activation of B cells has been improved can beexemplified. As a method for measuring/evaluating the alteration ofgeneration of antibody, for example, a method for measuring/evaluatingincrease or decrease of generation of antibody such as IgM antibody,IgG1 antibody, IgG2b antibody, IgG2a antibody in condition with andwithout the test substance to specify a substance in which generation ofantibody has been improved can be exemplified. As a method formeasuring/evaluating the alteration of production of cytokine, forexample, a method for measuring/evaluating increase or decrease ofproduction of cytokine such as interleukine-4, IFN-γ, interleukine-12 incondition with and without the test substance to specify a substance inwhich production of cytokine has been improved, can be exemplified.Further, as a method for measuring/evaluating the alteration of antigenpresentational ability in the antigen presenting cells, for example, amethod for measuring/evaluating increase or decrease of expressionamount of antigen specific to antigen presenting cells such as the majorhistocompatibility complex class II, CD80, CD86 in the antigenpresenting cells such as dendritic cells in condition with and withoutthe test substance to specify a substance in which antigenpresentational ability in the antigen presenting cells has improved, canbe exemplified. Though above-mentioned methods for measuring/evaluatingare not limited to these specifically, it is more preferable tocompare/evaluate the nonhuman model animal of Th2-mediated hyperimmuneresponse with the wild-type nonhuman animal of its littermate inscreening a therapeutic agent for an allergosis in respect of confirmingwhether there is a side-effect.

As candidate substances for an inducer/promoter or an inhibitor forTh2-mediated immune response or a promoter or an inhibitor for PIR-Afunction obtained from the method for screening an inducer/promoter oran inhibitor for Th2-mediated immune response or a promoter or aninhibitor for PIR-A function of the present invention, substances suchas prostaglandin E2 (PGE2), phospholipase A2 (PLA) can be exemplified.Further, as therapeutic agents for allergosis such as anaphylacticshock, bronchial asthma, and pollen allergen obtained from the methodfor screening a therapeutic agent for an allergosis of the presentinvention, therapeutic agents comprising the aforementionedinducer/promoter or the inhibitor for Th2-mediated immune response orthe inducer/promoter or the inhibitor for PIR-A function as effectiveingredients are exemplified, but they can be not limited to these.

The inducer/promoter or the inhibitor for Th2-mediated immune responseor the promoter or the inhibitor for PIR-A -function of the presentinvention obtained from the above-mentioned method for screening can beused for therapy for such as a patient requiring induction/promotion orinhibition of Th2-mediated immune response or promotion or inhibition ofPIR-A function, or a patient developing a disease by Th2-mediatedhyperimmune response such as allergosis. A therapeutic agent for anallergosis of the present invention can be administered orally orparenterally. For oral administration agent, it refers pharmaceuticalsolid such as powder, granule, capsule, and tablet or liquidpharmaceutical such as syrup and elixir, while for parenteraladministration agent, it refers injection, percutaneous pharmaceuticalor suppository. These formulations can be manufactured by addingauxiliaries accepted pharmacologically and pharmaceutically to activeingredients in a conventional manner. As auxiliaries, in oral agent andmucosal administration agent, ingredients for formulation, e.g. diluentssuch as light silicic acid anhydride, starch, lactose, crystallinecellulose, lactose calcium, disintegrant such as carboxymethylcellulose, lubricant such as magnesium stearate and others, ininjectable solution, ingredients for formulation e.g. solubilizer andauxiliary solubilizer agent such as saline, mannitol and propyleneglycolate, suspending agent such as surfactant and others, and further,in external preparation, ingredients for formulation, e.g. aqueous oroleaginous solubilizer or auxiliary solubilizer agent and adhesive areused. In addition, applied dose is suitably determined according to thekind of subjected disease, age, sex, weight, symptoms of a patient, anda dosage form.

The present invention will be explained more specifically with examplesbelow, but the technical scope of the invention is not limited to theseexamples.

EXAMPLE 1 Generation of PIR-B-Deficient Mice

Phage clones containing the 8.8 kb of mouse PIR-B gene were isolatedfrom the genomic library of 129/SvJ (Stratagene) with intracellularregion of B10A-derived PIR-B cDNA (1.0 kb of Eco RI-Xho I fragment) as aprobe. 5.0 kb of Eco RV-Eco RI fragment containing the region of exons1-7 and 5′ side of exon 8 was cut out and recloned into pBlueSK+cleavedwith Hin cII-Eco RI. This was cleaved with Xba I, filled up, and addedwith Sal I linker to make 5.0 kb of Xho I-Sal I fragment to be 5′-sidefragment. Another subclone containing exons 10-14 was used as atemplate, 2.0 kb of fragment was amplified by PCR using the primer addedwith Xho I linker on 5′-side (primer 1:5′-TATCCTCGAGCTTCTCCGACGAAGACATCG-3′; Seq. ID No. 1) and the primeradded with Sal I linker on 3′-side (primer 2:5′-AGATCGTCGACTGTTCAGTTGTTCCCTTGAC-3′; Seq. ID No. 2) to make 2.0 kb ofXho I-Sal fragment of post-cleavage of Xho I-Sal I was made to be3′-side fragment. 5.0 kb of 5′-side fragment and 2.0 kb of 3′-sidefragment were integrated into Xho I site and Sal I site of pMC1-Neovector which does not comprise Poly-A additional signal (pMC1-Neo-pA⁻;Stratagene), respectively. It was then cleaved with Xho I-Sal I, and 8.0kb fragment was integrated into the Xho I site of pIC19R-MC1tK vectorcontaining a herpes virus thymidine kinase to construct PIR-B targetingvector (FIG. 1 a; exons 9 and 10 are transmembrane regions). The vectorwas cleaved with Cla I site in the polylinker of the plasmid andlinearized.

The ES cell lines RW4 (10⁷ cells) maintained on feeder layer fromprimary embryonic fibroblasts (37° C., 5% CO₂) and 25 μg of PIR-Btargeting vector were suspended into 600 μl of phosphate buffered saline(PBS-), and electroporated at 800 V with 3 μF of capacitance using 4 cmwide cuvettes. After 10 min, the cells were infused separately into 60mm dishes by 10⁶ cells. After 48 h, selection was begun with 400 μg/mlof G418 and 0.2 μM of FIAU, the concentration was lowered to 300 μg/mlof G418 from the 5th day and to 225 μg/ml of G418 on the 7th day, G418was completely removed on the 8th day, and grown colonies (184 colonies)were picked up onto 96-well plates on the 10th day. Three daysthereafter, they were subcultured onto 24-well plates, further threedays thereafter, half of them were cryopreserved and the rest of themwere enlarged for DNA recovery, and two days later DNA was recovered.Homologous ES cell recombinants were obtained at a frequency of 2.2% (4cells) by Southern blotting analysis. The homologous ES cellrecombinants were injected into blastocysts to generate chimeric mice.The chimeric mice were confirmed by Southern blotting analysis with theuse of probes showing genomic DNA extracted from mouse splenic B cellsin FIG. 1 (FIG. 1 b). As a result, it was found that 4 male chimericmice had germline transferability. The above-mentioned male chimericmice were intercrossed with C57BL/6J female mice, heterozygous mice weregenerated from the littermates, and the heterozygous mice were thenintercrossed to generate homozygous mice at the expected Mendelianratios. Further, although the homozygous mice showed normal growth, whenPIR-B expression in splenic B cells of PIR-B-deficient mice was analyzedin protein level by immunoblotting, disappearance of PIR-B expressionwas confirmed (FIG. 1 c). Then, following examples were conducted withaforementioned PIR-B gene-deficient and wild-type mice at 6-32 weeksold.

It has been apparent that PIR-A and PIR-B co-express in a wide varietyof cells in the immune system such as B cells, mast cells (MCs),dendritic cells (DCs) and macrophages at least in mRNA level or reversetranscriptase-polymerase chain reaction (RT-PCR) level (Proc. Natl.Acad. Sci. USA. 94, 5261-5266, 1997; J. Exp. Med. 189, 309-318, 1999; J.Biochem. 123, 356-368, 1998). Flow cytometric analysis (J. Exp. Med.189, 309-318, 1999) of bone marrow-derived macrophages (BM-Macrophages)or bone marrow-derived dendritic cells (BM-DCs) using a monoclonalantibody 6C1 that recognizes a common epitope of PIR-A and PIR-B showedthe reduction of PIR expression in cells from PIR-B^(−/−) mice,suggesting that these cell types co-express PIR-A and PIR-B on theirsurfaces. That is, it is shown that PIR-A dominates PIR-B inBM-macrophages, whereas PIR-B dominates PIR-A in BM-DCs (FIG. 1 d). Onthe other hand, analysis of BM-MCs and splenic B cells revealed thatPIR-B expresses exclusively on these cell surfaces because the PIRstaining was almost gone in the cells from PIR-B^(−/−) mice (FIG. 1 d).The aforementioned results in normal cells and cells from PIR-B^(−/−)mice elucidated respective expression profiles of PIR-A and PIR-B oneach cell in immune system, and these results and description inarticles (Proc. Natl. Acad. Sci. USA. 94, 5261-5266, 1997; J. Biochem.123, 358-368, 1998) suggested the importance of PIR-A/B expressionbalance on each immune systemic cell for immune regulation. On the otherhand, expression balances of murine IgLR, gp49A and gp49B are known tobe specific according to cell-types (Mol. Cell. Biol. 20, 7178-7182,2000).

EXAMPLE 2 Flow Cytometric Analysis of B Cell Development in Wild-Typeand PIR-B^(−/−) Mice

Splenic CD4/CD8 T cells and T/B cell population ratios were notsignificantly changed in PIR-B^(−/−) mice, suggesting that PIR-Bdeficiency does not influence general T cell development. On the otherhand, Kubagawa et al. has reported that the expression of PIR moleculesis regulated in each process of B cell development, maturation andactivation (J. Exp. Med. 189, 309-318, 1999). It is reported that PIRexpression levels are highest on marginal zone B cells, and they arehigher in the peritoneal B-1 B cells than those in B-2 B cells (J. Exp.Med. 189, 309-318, 1999). Then, examination of the expression ofmolecule in each cell from PIR-B^(−/−) mice showed a normal profile inexpression of B220 and IgM in bone marrow cells from PIR-B^(−/−) mice(FIG. 2 a). When splenic B cells from PIR-B^(−/−) mice were comparedwith those from wild-type, it was confirmed that splenic B cells fromPIR-B^(−/−) mice contained IgM^(l0) and IgD^(hi) of mature recirculatingfollicular B cells and IgM^(hi) IgD^(hi) of type 2 transitional B cells(T-2 B cells) equivalent to those from wild-type, but slightlyincreasing in type 1 transitional B cells (T-1 B cells) of immature typeIgM^(hi) IgD^(l0) (FIG. 2 b). CD23⁻CD21⁺ marginal zone B cells,CD23⁺CD21⁺ follicular B cells and CD23⁻CD21⁻ B cells in B220⁺ spleniccells could be observed in PIR-B^(−/−) mice at levels comparable tothose of wild-type mice (FIG. 2 c).

Meanwhile, CD5⁺IgM⁺ peritoneal B-1 cells were more significantlyincreased in PIR-B^(−/−) mice compared with those of wild-type mice,[FIG. 2 d; Mean values % ±SD of B-1 cells were 23.0±4.8 (n=3) inwild-type mice, 36.4±5.6 (n=4) in PIR-B^(−/−) mice, p<0.05, see alsoFIG. 3 b], indicating that the development or increase of the B-1 cellpopulation is promoted by PIR-B deficiency. It is reported that the B-1cells produce natural antibodies and play an important role inautoimmunity (Annu. Rev. Immunol. 19, 595-621, 2001; Nature 357, 77-80,1992). Increased B-1 cells are also observed in mice which are deficientin either CD22 (Science 274, 798-801, 1996; Nature 384, 634-637, 1996)or CD72 (Immunity 11, 495-506, 1999), both of which have ITIM(s).However, significant increase of IgM amount in serum could not bedetected in PIR-B^(−/−) mice at 6 or 32 weeks of age. In addition,anti-double stranded DNA antibodies could not be detected at least up to32 weeks of age in PIR-B^(−/−) mice in contrast to CD22-deficient mice,in which hyper IgM and autoantibody production are evident at the samedevelopment stage (Science 274, 798-801, 1996; Nature 384, 634-637,1996).

EXAMPLE 3 B Cell Proliferation Response of PIR-B-Deficient Mice to BCR

Ho et al. has reported that irrespective of the cell activation status,PIR-B molecules in microphages and B cells were constitutivelyphosphorylated, and PIR-B in splenocytes were constitutively associatedwith the SHP-1 and Lyn (Proc. Natl. Acad. Sci. USA. 96, 15086-15090,1999). In Lyn-deficient mice, PIR-B tyrosine phosphorylation was greatlyreduced (Proc. Natl. Acad. Sci. USA. 96, 15086-15090, 1999) and PIR-Bligation on chicken DT40 cells inhibited the BCR-induced tyrosinephosphorylation of Iga/Igβ, Syk, Btk and PLC-γ2 (Oncogene 18, 2291-2297,1999) were reported. Further, it was revealed that the deletion of PIR-Binduces hypersensitive B cells upon BCR ligation from the constitutiveassociation of SHP-1 with PIR-B, as seen in CD22-deficient mice (Science274, 798-801, 1996; Nature 384, 634-637, 1996). Here, the presentinventors analyzed the proliferative response of splenic B-2 cells uponIgM F (ab)₂ antibody stimulation and found a significantly enhancedproliferation of the B-2 cells from PIR-B^(−/−) mice (FIG. 3 a). Theenhanced proliferation was more significant by masking an inhibitoryeffect by FcγRIIB which is an unique inhibitory Fcγ receptor on B cells,when stimulated with rabbit IgG anti-IgM antibody (Immunity 3, 635-646,1995; Nature 379, 346-349, 1996) (FIG. 3 b). Injecting wild-type orPIR-B^(−/−) mice with anti-IgM F (ab′) ₂ antibody intraperitoneally andanalyzing with flow cytometry, the peritoneal B-1 cells from PIR-B^(−/−)mice showed remarkable proliferation, whereas those from wild-type micedid not show proliferation excessively (FIG. 3 c). Since an inhibitoryregulator of the response raised by BCR crosslinking in B-1 cells wasreported (Science 274, 1906-1909, 1996), in order to examine whether thecell proliferative response raised on co-crosslinkage of BCR increasesin PIR-B^(−/−) mice, the cells were stimulated with soluble F (ab′)₂ IgMantibody, anti-CD40 antibody (CD40), or LPS and examined theproliferation of splenic B220 positive cells from uptake amount of[³H]-thymidine. It revealed that PIR-B^(−/−) cells become hypersensitivestatus to proliferate by stimulation via ligation with BCR. However, asa result of ligation of splenic B-2 cells from wild-type mice with BCRin vitro, a significant increase of PIR-B expression into thedetergent-insoluble membrane fraction could not be observed. It hasshowed that there is no constitutive association of PIR-B with BCR.Therefore, it is probable that the PIR-B may not be a specificinhibitory co-receptor for BCR. Rather, PIR-B may down-regulate BCRsignaling by interacting with any ligand, and B cells might obtainhypersensitivity to stimulation via BCR by PIR-B deficiency generally.

EXAMPLE 4 Immune Response and Proliferation of Lymph Node Cells inPIR-B-Deficient Mice

Then, PIR-B^(−/−) mice were injected with TNP-Ficoll or TNP-LPS as TIantigens to observe the immune responses to various substances fromabove-mentioned results. The result revealed that anti-TNP IgM antibodyresponses to respective substances showed significant increases inPIR-B^(−/−) mice compared to those in wild-type mice (FIG. 4 a, 4 b),being consistent with the results of hypersensitive B cells inPIR-B^(−/−) mice (see Example 3). In order to examine whether PIR-Bdeficiency influences acquired immune response in vivo, wild-type miceor PIR-B-deficient mice were immunized with TD antigen (TNP-KLH+CFAadjuvant or TNP-KLH+pertussis toxin+alum adjuvant) and antibody titer inserum was measured. The results are shown in FIG. 5 a. In immunizationwith TNP-KLH+pertussis toxin+alum adjuvant initiating Th2-mediatedresponse, compared PIR-B-deficient mice with wild-type mice, it wasconfirmed that the former showed more increased anti-TNP IgG1 antibodytiter or anti-TNP IgM antibody titer at primary immunization responseand increased anti-TNP IgG1 antibody or anti-TNP IgG2b antibody anddecreases anti-TNP IgG2a antibody titer at secondary immunization thanthe latter (FIG. 5 a). As mentioned above, the increase of IgG1 antibodytiter induced by Th2-mediated cytokine and decrease of IgG2a antibodytiter induced by Th1 type cytokine in PIR-B-deficient mice suggestedthat PIR-B might affect the differentiation of T cells into Th1/Th2 inimmune response to TD antigens in vivo. There, in order to examine theeffect of PIR-B deficiency on activation, differentiation andproliferation of T cells in vivo, wild-type or PIR-B-deficient mice wereimmunized with TNP-KLH or OVA (ovalbumin) onto their footpads, andproduction and proliferation of cytokine were measured when lymph nodecells from PIR-B^(−/−) mice isolated 11 days later from the immunizationwere re-stimulated with antigen. The results are shown in FIG. 5 b. Itshowed that IL4 (interleukin 4) production was promoted while IFN-γproduction was inhibited, but significant difference could not be foundin cell proliferation in PIR-B deficient mice. This suggested thatalthough the difference of activation and proliferative ability of Tcells could not be found in wild-type and PIR-B-deficient mice, T cellsfrom PIR-B-deficient mice are prone to Th2-type. This result was notinconsistent with the result that IgG1 increased and IgG2a decreased inPIR-B as seen in generation of antigen.

EXAMPLE 5 Lack of Maturation of PIR-B^(−/−) Mice and Decrease of IL-12Production

In order to explore how the mechanism for the Th2-exaggerated responsesin PIR-B^(−/−) mice was, bone marrow dendritic cells (BM-DCs) wereisolated from wild-type or PIR-B^(−/−) mice, the cells were pulsed withOVA, and DCs surface markers were analyzed by flow cytometry before andafter the pulse with OVA. The results are shown in FIG. 6 a. Thisrevealed that DCs from PIR-B^(−/−) mice were relatively immature.Expression ability of MHC class II before the antigen pulse was lower inDCs from PIR-B-/- mice than those from wild-type mice (FIG. 6 a), whileCD80 and CD86 expression were comparative in the DCs from PIR-B^(−/−)mice and from wild-type. DCs from wild-type mice after pulse with OVAwere confirmed to be well matured and concomitantly the expression ofboth MHC class II and CD80/86 were enhanced while the enhancedexpression of these molecules on DCs from PIR-B^(−/−) mice was minimal.In addition, further stimulation with anti-CD40 antibody failed toimprove the relatively immature status of DCs from PIR-B^(−/−) mice. Ithas been reported that the above-mentioned immature DCs are prone toinducing Th2-dediated response (J. Immunol. 167, 1982-1988, 2001). Then,examination of the production of cytokine IL-12 (Immunity 4, 471-481,1996) which strongly induced Th1 revealed that generation of IL-12induced with OVA stimulation greatly diminished DCs from PIR-B^(−/−)mice (FIG. 6 b). Moreover, the amount of FITC-labeled OVA incorporatedinto the DCs could be observed to be comparable between the cells fromwild-type and those from PIR-B^(−/−) mice (FIG. 6 c). From aboveresults, it seems that the degree of the activation-dependant maturationof DCs upon antigen uptake is irrelevant with PIR-B deficiency.Therefore, it can be thought that PIR-B deficiency restricts DCs in itsrelatively immature status even when sufficient amount of antigenstimulation is added to the cells.

IgLRs, an immunoreceptor family of cell surface molecules that may playregulatory roles in cellular signaling and in immune responses (Science290, 84-89, 2000; Activating and Inhibitory Immunoglobulin-likeReceptors, 7-15, 2001; Annu. Rev. Immunol. 17, 875-904, 1999) is knownto include such as human killer cell IgLR, Ig-like transcript/leukocyteIgLR/myeloid IgLR (ILT/LIR/MIR), leukocyte-associated IgLR (LAIR),signal induction receptor proteins or SIRP both in mouse and human,murine gp49 molecules and PIR. Each molecule of human ILT/LIR/MIR isconsidered to be closest relatives of murine PIR (J. Biochem. 123,358-368, 1998; Activating and Inhibitory Immunoglobulin-like Receptors,7-15, 2001; Proc. Natl. Acad. Sci. USA. 21, 13245-13250, 2000). Thepresent invention revealed that PIR-B has physiological role tonegatively regulate the peritoneal B-1 cells, B cell sensitivity to BCRstimulation, humoral response to TI antigen, Th2 response to TD antigenand the like. Although it is suggested that MHC class I molecule mayinteract with PIR based on the report that PIR phosphorylation wasdecreased in B2-microgloblin-deficient mice (Proc. Natl. Acad. Sci. USA.96, 15086-15090, 1999), attempts to identify the PIR-specific ligandhave not been successful yet. The present invention can not only analyzehow the activation threshold for B cells is determined by PIR-B but alsoelucidate the ligand for PIR-B based on the result from consideration ofB cells and DCs. Specifically, ligand can be identified by preparingfusion protein of PIR and examining interaction with the targeted cells.Further, in PIR, the molecular mass of protein from targeted cells isidentified by Western blotting analysis, the candidate protein isprepared when the protein is a known molecule and the ligand can beidentified by determining the ligation with PIR, because there is highpossibility for expression of ligand on T cells.

Experimental Method

Method 1 (Immunobot Analysis)

B220 positive splenic B cells were selected from splenic B cells (10⁸)by MACS sorting using B220-beads (Miltenyi Biotec) and lysed. Proteinscontained in the supernatants of the cell-lysates were separated bySDS-PAGE using a 7.5% gel and transferred onto a PVDF (ImmobilonP,Millipore) membrane. The membrane was incubated under coexistence ofgoat anti-PIR-A/B antibody (Santa Cruz Biotech), followed by labelingwith biotin-linked monkey anti-goat Ig antibody andperoxidase-conjugated streptavidin (Amersham Pharmacia) as the secondaryantibodies and visualization.

Method 2 (Antibodies and Flow Cytometry)

Flow cytometric analysis was performed with the use of monoclonalantibodies (all the antibodies used were products of BD PharMingen) suchas FITC-, PE-, or biotin-conjugated anti-mouse IgM antibody (R6-60.2),anti-mouse IgD antibody (11-26), anti-mouse CD5 antibody (Ly-1),anti-mouse B220 antibody (RA3-6B2), anti-mouse CD21 antibody (7G6),anti-mouse CD23 antibody (B3B4), anti-c-kit antibody (2B8), anti-CD11bantibody (M1/70), anti-CD11c antibody (HL3), anti-CD80 antibody(16-10A1), anti-CD86 antibody (GLI), CD40 (3/23), anti-MHC class IIantibody (M5/114.15.2) and the like. TRI color-conjugated streptavidin(Caltag) was used for staining biotin-antibody. 6C1 of anti-PIRmonoclonal antibodies (J. Exp. Med. 189, 309-318, 1999) were provided byDrs. M. D. Cooper and H. Kubagawa (University of Alabama). IgM F (ab′)₂antibodies were purchased from ICN Laboratories. Cell surface stainingwas carried out according to the method publicly known and flowcytometric analysis was performed with a FACSCalibur using CellQuestsoftware (Becton Dickinson). Dead cells were eliminated from theanalysis, and measurement was performed on the basis with propidiumiodide staining.

Method 3 (Proliferation Response of B Cells)

Splenic B220 positive cells were purified by MACS (Mylteni). The cellswere activated with soluble anti-IgM F (ab′)₂ antibody, anti-CD40antibody (CD40) or LPS, and 48 h after the stimulation, proliferation ofsplenic B220 positive cells was determined by [³H]-thymidine uptake. Forproliferation of peritoneal B-1 cells, wild-type or PIR-B^(−/−) micewere intraperitoneally injected with 200 μg of soluble anti-IgM F (ab′)₂antibody, respectively and determined in the same manner as theabove-mentioned. After 48 h, peritoneal cells from the individual mousewere stained with biotinylated anti-mouse CD5 monoclonal antibody addedwith phycoerythrin-conjugated streptavidin or FITC-conjugated anti-mouseIgM monoclonal antibody, and the ratio of B-1 (CD5 positive IgMpositive) cell populations was analyzed.

Method 4 (Humoral Response and T Cell Proliferation)

Wild-type or PIR-B-gene-deficient mice were intraperitoneally injectedwith 100 μg of TNP-Ficoll, 50 μg of TNP-LPS, or with OVA lysed in 10 μgof TNP-KLH or aluminium hydroxide to be immunized. The mice werere-stimulated with antigen after 8 weeks of injection of TD antigen toinduce the secondary response, and the antibody titer to TNP in serumwas determined by ELISA with absorbance at 450 nm. Lymph node cells werecollected from popliteal lymph nodes of wild- type and PIR-B^(−/−) mice11 days after from the primary immunization with TNP-KLH or OVA. Thecollected cells were activated with either the same antigen asabove-mentioned one or T cell mitogen, 40 h after the stimulation, thesupernatant was harvested and the yield of IL-4 or IFN-γ in the culturesupernatant was determined by ELISA. T cell proliferation was determinedby [³H]-thymidine uptake on the 3rd day of the culture.

Method 5 (DC Maturation and Antigen Uptake)

In order to examine BM-DCs, BM cells from wild-type or PIR-B^(−/−) micewere cultured in the presence of recombinant mousegranulocyte-macrophage colony stimulating factor (GM-CSF) (PeproTech),respectively medium was changed on the 4th day, and the cells werefurther cultured. On the 6th day of the culture, it was observed thatthe above BS cells expressed MHC class II molecules, CD40, CD80, CD86and CD11c and became immature DCs. In order to mature the DCs, 50 μg/mlof OVA or 1 μg/ml of anti-CD40 antibody (BD Pharmigen) was added to DCculture 24 h before the harvest of the cells for stimulation. Afterstimulation, culture supernatants of DC culture were harvested and mouseIL-12 p70 in the supernatants was measured by ElISA (BD Pharmingen). Theabove cells were then stained with CD80, CD86 and anti-MHC class IIantibody as mature cell markers, and analyzed by flow cytometry. So asto measure the amount of antigen uptake by DCs, the DCs were incubatedunder coexistence of 20 μg/ml of FITC-conjugated OVA (Molecular Probes)at 37° C. or 4° C. for 3 h, then washed and stained with phycoerythrin(PE) -conjugated anti CD11c antibody. Uptake amount of FITC-OVA by DCsat 37° C. was measured by flow cytometry.

Industrial Applicability

A nonhuman model animal of Th2-mediated hyperimmune response of thepresent invention allows to analyze the Th2-mediated immune responsemechanism and allergy onset mechanism in vivo therewith, and it isuseful in studying an allergy onset process as well as a hyperimmuneresponse of B cells, and the use of the nonhuman model animal ofTh2-mediated hyperimmune response makes it possible to screentherapeutic agents for allergosis such as anaphylactic shock, bronchialasthma, and pollen allergen and to provide the method for treatingallergosis.

1. A nonhuman model animal of Th2-mediated hyperimmune response whichinduces hyper-response of a B cell and facilitates development of anallergy by lacking PIR-B gene function on its chromosome.
 2. Thenonhuman model animal of Th2-mediated hyperimmune response according toclaim 1, wherein it develops the allergy more excessively than awild-type nonhuman animal by immunizing an adjuvant that initiatesTh2-mediated response.
 3. The nonhuman model animal of Th2-mediatedhyperimmune response according to claim 2, wherein the adjuvant thatinitiates Th2-mediated response is an alum adjuvant comprising a TNP-KLHand a pertussis toxin.
 4. The nonhuman model animal of Th2-mediatedhyperimmune response according to any one of claims 1 to 3, wherein thenonhuman animal is a mouse.
 5. A method for screening aninducer/promoter or an inhibitor for Th2 -mediated immune response or apromoter or an inhibitor for PIR-A function, wherein an alteration ofTh2-mediated immune response or PIR-A function is determined/evaluatedby administering a test substance to the nonhuman model animal ofTh2-mediated hyperimmune response according to any one of claims 1 to 4or contacting a tissue, an organ or a cell derived from the animal tothe test substance.
 6. The method for screening an inducer/promoter oran inhibitor for Th2-mediated immune response or a promoter or aninhibitor for PIR-A function according to claim 5, wherein thealteration of Th2-mediated immune response is an alteration ofactivation of the B cell, generation of antibody, production of cytokineand/or antigen presentational ability in an antigen presenting cell. 7.The method for screening an inducer/promoter or an inhibitor forTh2-mediated immune response or a promoter or an inhibitor for PIR-Afunction according to claim 6, wherein the alteration of activation ofthe B cell is an alteration of development or proliferation of the Bcell.
 8. The method for screening an inducer/promoter or an inhibitorfor Th2-mediated immune response or a promoter or an inhibitor for PIR-Afunction according to claim 6, wherein the alteration of generation ofantibody is an alteration of generation of an IgM antibody, an IgG1antibody, an IgG2b antibody and/or an IgG2a antibody.
 9. The method forscreening an inducer/promoter or an inhibitor for Th2-mediated immuneresponse or a promoter or an inhibitor for PIR-A function according toclaim 6, wherein the alteration of production of cytokine is analteration of production of interleukin-4, IFN-γ and/or interleukin-12.10. The method for screening an inducer/promoter or an inhibitor forTh2-mediated immune response or a promoter or an inhibitor for PIR-Afunction according to claim 6, wherein the alteration of antigenpresentational ability in the antigen presenting cell is an alterationof expression amount of a major histocompatibility complex class II,CD80 and/or CD 86 in a dendritic cell.
 11. The method for screening aninducer/promoter or an inhibitor for Th2 -mediated immune response or apromoter or an inhibitor for PIR-A function according to any one ofclaims 5 to 10, wherein a case with the nonhuman model animal ofTh2-mediated hyperimmune response according to any one of claims 1 to 4is compared/evaluated with a case with the wild-type nonhuman animal.12. A method for screening a therapeutic agent for an allergosis,wherein the alteration of Th2-mediated immune response or PIR-A or PIR-Bfunction is determined/evaluated by administering a Th2-mediated immuneresponse initiating adjuvant and the test substance to the nonhumanmodel animal of Th2-mediated hyperimmune response according to any oneof claims 1 to 4, or contacting the tissue, the organ or the cellderived from the animal to the Th2-mediated immune response initiatingalum adjuvant and the test substance.
 13. The method for screening atherapeutic agent for an allergosis wherein the alteration ofTh2-mediated immune response is an alteration of activation of the Bcell, generation of antibody, production of cytokine and/or antigenpresentational ability in the antigen presenting cell.
 14. The methodfor screening a therapeutic agent for an allergosis according to claim13, wherein the alteration of the B cell is an alteration of developmentor proliferation of B the cell.
 15. The method for screening atherapeutic agent for an allergosis according to claim 13, wherein thealteration of generation of antibody is an alteration of generation ofan IgM antibody, an IgG1 antibody, an IgG2b antibody and/or an IgG2aantibody.
 16. The method for screening a therapeutic agent for anallergosis according to claim 13, wherein the alteration of productionof cytokine is an alteration of production of interleukin-4, IFN-γand/or interleukin-12.
 17. The method for screening a therapeutic agentfor an allergosis according to claim 13, wherein the alteration ofantigen presentational ability in the antigen presenting cell is analteration of expression amount of the major histocompatibility complexclass II, CD80 and/or CD 86 in the dendritic cell.
 18. Aninducer/promoter or an inhibitor for Th2-mediated immune response or apromoter or an inhibitor for PIR-A function obtained by the method forscreening an inducer/promoter or an inhibitor for Th2-mediated immuneresponse or a promoter or an inhibitor for PIR-A or PIR-B functionaccording to any one of claims 5 to
 11. 19. The inducer/promoter or theinhibitor for Th2-mediated immune response or the promoter or theinhibitor for PIR-A function according to claim 18, wherein theinducer/promoter or the inhibitor for Th2-mediated immune response is atherapeutic agent for a disorder attributed to Th2-mediated hyperimmuneresponse.
 20. The inducer/promoter or the inhibitor for Th2-mediatedimmune response or the promoter or the inhibitor for PIR-A functionaccording to claim 19, wherein the disorder attributed to Th2-mediatedhyperimmune response is an allergosis.
 21. A therapeutic agent for anallergosis obtained by the method for screening a therapeutic agent foran allergosis according to any one of claims 12 to
 17. 22. A method fordiagnosis of an allergosis wherein a disease symptom of the nonhumanmodel animal of Th2-mediated hyperimmune response according to any oneof claims 1 to 4 is utilized therefor.